少加样高收效制备创伤修复剂纤维粘连蛋白的方法(英文)  

Study on the preparation method for lower-sampling and large-scale purification of a wound-healing agent: fibronectin

作　　者：谭见容[1] 任国梅[1] 戴丽冰[1] 黄雪芳[1] 

机构地区：[1]广州市红十字会医院暨南大学第四附属医院创伤外科研究所,广东省广州市510220

出　　处：《中国临床康复》2005年第47期167-169,共3页Chinese Journal of Clinical Rehabilitation

摘　　要：背景纤维粘连蛋白不仅对细胞起支架作用,而且是细胞间重要的连结因子,具有类似调理素作用,可促进单核吞噬细胞系的吞噬功能,在炎症和创伤中起修复作用。目的以新鲜健康男性“O”型血浆为原料,为制备纤维蛋白结合素筛选出加样量小,方法简便、收得率高的纯化方法。设计开放性实验。单位广州市红十字会医院暨南大学第四附属医院创伤外科研究所。材料实验于2000-07/2002-07在广州市红十字会医院创伤外科研究所完成。材料主要包括新鲜健康男性“O”型血浆、明胶、CNBr-activatedSepharose4B、交联葡聚糖G-25、尿素、三羟甲基氨基甲烷等。方法用明胶与溴化氰活化琼脂糖珠偶联的亲和层析柱对纤维蛋白结合素进行纯化。①加人血浆取新鲜健康男性O型血浆150m L,每次向柱内加入25m L,停留20min,流速3.5m L/m in。②去杂蛋白用pH7.5的Tris-柠檬酸钠平衡液洗柱,洗至流出液280nm下吸光度值<0.02,再用含1mol/L NaCl的Tris-柠檬酸钠洗去其他杂蛋白。③收集纤维蛋白结合素用3m ol/L脲-Tris洗脱液洗柱,收集纤维蛋白结合素。④纤维蛋白结合素收集液去尿素取纤维蛋白结合素的收集液,过SephadeG-25,去除尿素。⑤除菌用0.22μm滤菌器过滤除菌。主要观察指标①十二烷基黄酸钠-聚丙烯酰胺凝胶电泳结果。②停留时间对提纯纤维蛋白结合素收得率的影响。③加入血浆量对提纯纤维蛋白结合素收得率的影响。结果①十二烷基黄酸钠-聚丙烯酰胺凝胶电泳结果分离胶浓度为7%,浓缩胶浓度为3%,纤维蛋白结合素电泳结果为单一蛋白区带。②停留时间对提纯纤维蛋白结合素收得率的影响血浆加样量为150m L,柱内不停留和停留时间为10,20,30m in时纤维蛋白结合素收得率分别是(65.24±3.45)%,(74.77±4.05)%,(86.99±4.10)%,(80.47±3.75)%。③加入血浆量对提纯纤维蛋白结合素收得率的影响柱内停留时间为20m iBACKGROUND： Fibronectin serves not only as the supporter for cells, but also as an important intraceUular linkage, possessing opsonic-like functions. It can promote the phagocytic function of mononucleophages and the repair in inflammation and trauma. OBJECTIVE： Type O plasma was freshly obtained from healthy males in search of the optimal preparation method for lower sampling, convenient and higher-yielding of fibrenectin. DESIGN： Open experiment. SETTING： Institute of Traumatic Surgery, Fourth Affiliated Hospital of Guangzhou Red Cross Hospital, Jinan University. MATERIALS： This experiment was carded out in the Institute of Traumatic Surgery of Guangzhou Red Cross Hospital between July 2000 and July 2002. The major materials consisted of type 0 fresh plasma from healthy males, gelatin, sepharose 4B activated with cyanogen bromide, sephedex G-25, urea, and trishydroxymethylaminomethane. METHODS： Affinity column constituted by gelatin coupling with sepharose 4B activated with cyanogen bromide was used to purify flbronectin. （1） Human plasma was added： 150 mL type 0 plasma was freshly obtained from healthy males and added into the column once by 25 mL at an interval d 20 minutes, the speed of flow was 3.5 mL/min. （2） Removing mixed protein： the column was rinsed by Tris-sedium citrate equilibrium liquid （pH 7.5） until the absorbency of flow-ont fluid decreased to 〈 0.02 at 280 nm. Again lmol/L NaC1 containing Tris-sodium citrate was used to wash off other mixed proteins. （3） Collecting fibronectifi： the colonto was rinsed by urea-Trispurge Fluid （3 tool/L） to collect fibronectin. （4） Removing urea from fibronectin collection： fihronectin collection liquid was filtrated by Sephade G-25 to remove urea. （5）Disinfection： 0.22 pan germtight filter was used for disinfection. MAIN OUTCOME MEASURES ： （1）Results of sodium dodecyl benzene sulfonate-polyacrylamide gel electrophoresis. （2） The influence of retention time on the yield of purified fibronectin. （3�

关 键 词：纤维蜜白 结合素类/生物合成 纤连蛋白类/生物合成 

分 类 号：R318[医药卫生—生物医学工程]